【摘要】 为快速检测家畜嗜衣原体,选取家畜嗜衣原体主要外膜蛋白(MOMP)基因的高度保守区域,设计特异性引物和探针,通过对反应条件的优化,建立家畜嗜衣原体TaqMan-MGB荧光定量PCR检测方法。结果显示,该方法的最低检测限为2.8×10copies/μL,在2.8×102~2.8×106 copies/μL范围内具有良好的线性关系,其标准曲线方程为y=-3.281 5x+41.396,线性相关系数r2=0.999 4,扩增效率为101.7%;该方法可特异性检测家畜嗜衣原体,对肺炎嗜衣原体、沙眼衣原体、鹦鹉热嗜衣原体、流产嗜衣原体等6种动物衣原体无交叉反应,特异性好;其批间重复试验和批内重复试验的变异系数分别为0.170 2%~0.467 2%、0.331 4%~2.796%,重复性良好。采用本试验建立的方法与OIE推荐的方法进行比较,二者的结果表现出良好的符合率。该方法的建立为家畜嗜衣原体的早期检测和流行病学调查提供了有效的检测手段。更多还原

【Abstract】 In order to detect rapidly Chlamydophila pecorum,primers and specific TaqMan-MGB hybridization probes targeting major outer membrane protein(MOMP)gene of C.pecorum were designed.Through the optimization of reaction conditions,the detection method of real-time PCR with TaqMan-MGB for C.pecorum was established.The result shows that the detection limit of the assay was 2.8×10copies/μL.The standard curve was linear in the concentration range from 2.8×102 to 2.8×106copies/μL.The calibration curve equation is y=-3.281 5 x+41.396,the correlation coefficient is r2=0.999 4,and the expanding efficiency rate is 101.7%.The method can only detect C.pecorum and has no cross reaction with Chlamydophila pneumomiae,Chlamydia trachomatis,Chlamydophila psittaci,and Chlamydophila abortus,respectively.The coefficients of variation within batch and among batch are from 0.170 2%to 0.467 2%,and from 0.331 4%to 2.796%respectively.Compared real-time PCR with the method recommended by OIE,the coincidence rate is high,and it provides the effective detection method to the possibility of early detection and epidemiological surveys for C.pecorum.更多还原